The overall objective of this proposal is to understand the cellular and molecular mechanisms subserving the endocytosis and metabolism of insulin- receptor complexes and to then determine the biological significance of these processes in insulin action. Further understanding in this area is essential for the eventual elucidation of the pathophysiology of insulin resistant states such as obesity and type II diabetes mellitus. Although receptor-mediated internalization and intracellular processing of insulin-receptor complexes are fundamental properties in all insulin target cells, at present neither the molecular mechanism nor the functional roles of these processes are well understood. The work accomplished thus far has led to identification of the general region of the human insulin receptor (hIR) intracytoplasmic domain that is important for endocytosis. Accordingly, the objective of this proposal is to contribute to further understanding in this area by pursuing the following specific aims: 1) to identify and characterize the amino acid residues (internalization domain) in the intracytoplasmic juxtamembrane region of hIR which mediate its endocytic function. 2) To determine the relationship of the internalization domain to other functional domains of the hIR beta-subunit. 3) To assess the role of the internalization of insulin-receptor complexes in insulin action using mutant hIR with altered endocytic properties. 4) To generate and characterize antipeptide monoclonal antibodies directed against the internalization domain of hIR; and 5) to use the monoclonal antibodies to explore the role of internalization in insulin action in different insulin target cells. In pursuing these aims, extensive cellular, biochemical and molecular biological methodologies will be utilized. In brief these will include cell culture, DNA cloning, mutagenesis, transfection and generation of hIR mutants, generation of monoclonal antibodies and various functional studies including endocytosis and processing of hIRs as well as measurement of several insulin-stimulated biological actions. These will include: stimulation of glucose uptake (a rapid transmembrane effect); stimulation of glycogen and protein synthesis (effects of intermediate duration); and stimulation of RNA and DNA synthesis (longer-term effects). It is expected that these studies will provide novel information that will lead to a better understanding of the mechanism of insulin action and thereby contribute to the eventual elucidation of the pathophysiology of insulin resistance, a major abnormality in obesity and type II diabetes mellitus.